Recent advances in CE and microchip-CE in clinical applications: 2014 to mid-2017.

CE and microchip CE (ME) are powerful tools for the analysis of a number of different analytes and have been applied to a variety of clinical fields and human samples. This review will present an overview of the most recent applications of these techniques to different areas of clinical medicine during the period of 2014 to mid-2017. CE and ME have been applied to clinical chemistry, drug detection and monitoring, hematology, infectious diseases, oncology, endocrinology, neonatology, nephrology, and genetic screening. Samples examined range from serum, plasma, and urine to lest utilized materials such as tears, cerebral spinal fluid, sweat, saliva, condensed breath, single cells, and biopsy tissue. Examples of clinical applications will be given along with the various detection systems employed.

Detection of cerebral spinal fluid-associated chemokines in birth traumatized premature babies by chip-based immunoaffinity CE.

A major concern in treating premature infants with birth-associated head trauma is the rapid determination of reliable biomarkers of neuroinflammation. To this end a chip-based immunoaffinity CE device has been applied to determine the concentrations of inflammation-associated chemokines in samples of cerebral spinal fluid collected from such subjects. The chip utilizes replaceable immunoaffinity disks, to which reactive antibody fragments (FAb) of six antichemokine-specific antibodies were immobilized. Following injection of a sample into the device, the analytes were captured by the immobilized FAbs, labeled in situ with a red laser dye, chemically released and separated by CE. Each resolved peak was measured on-line by LIF detection and the results compared to standard curves produced by running known chemokine standards through the immunoaffinity system. The complete processing of a sample took 10 min with separation of all six analytes being achieved in less than 2 min. The system compared well to commercial ELISA, analysis of the results by linear regression demonstrating r(2) values in the range of 0.903-0.978, and intra and interassay CV of the migration times and the measured peak areas being less than 2.3 and 5%, respectively. Application of the system to analysis of cerebrospinal fluid from head traumatized babies clearly indicated the group with mild trauma versus those with severe injury. Additionally, CE analysis demonstrated that the severe trauma group could be divided into individuals with good and poor prognosis, which correlated with the clinical finding for each patient.

Assessment of chemokine profiles in human skin biopsies by an immunoaffinity capillary electrophoresis chip.

Atopic dermatitis is a skin condition resulting in a skin rash from exposure to environmental factors. Skin biopsies taken from patients suffering from atopic dermatitis were micro-dissected and analyzed using a microchip-based immunoaffinity CE system for the presence of CXCL1, CXCL5 and CXCL8 and CCL1, CCL3 and CCL5 chemokines. Disposable immunoaffinity disks with immobilized antibodies were used to capture the CXC and CC chemokines from the homogenized skin samples. The captured analytes were then labeled with AlexaFluor 633, eluted from the disk and separated by CE. The labeled chemokines were identified and quantified by laser induced fluorescence. The total analysis time was less than 40min, including the biopsy microdissection, pre-analysis preparation of the sample and the ICE-CHIP analysis, which took less than 10min with inter- and intra-assay CV's below 6.4%. Microchip-based immunoaffinity CE could distinguish between normal skin biopsies and those with inflammation. Patients with neutrophil cellular infiltrates by histopathology showed increased concentrations of CXCL1, CXCL5 and CXCL8 while increases of CCL1, CCL3 and CCL5 corresponded to the patient group demonstrating monocytic and T-lymphocyte infiltration by histopathology. This system demonstrates the ability to identify and quantify immunochemical analytes in frozen sections taken from clinical histopathology samples.

Psychological hardiness predicts neuroimmunological responses to stress.

Psychological hardiness characterizes people who remain healthy under psychosocial stress. The present exploratory study investigates possible links between hardiness and several immune and neuroendocrine markers: IL-6, IL-12, IL-4, IL-10, & neuropeptide-Y. A total of 21 Norwegian navy cadets were studied in the context of a highly stressful military field exercise. Blood samples were collected midway, and again late in the exercise when stress levels were highest. Psychological hardiness (including commitment, control, and challenge) was measured two days before the exercise. While all subjects scored high in hardiness, some were high only in commitment and control, but relatively low in challenge. These "unbalanced" hardiness subjects were also more stress reactive, showing suppressed proinflammatory cytokines (IL-12), increased anti-inflammatory cytokines (IL-4, IL-10), and lower neuropeptide-Y levels as compared to the hardiness-balanced group. This study thus shows that being high in hardiness with a balanced profile is linked to more moderate and healthy immune and neuroendocrine responses to stress.

Stromal factors SDF1α, sFRP1, and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells.

Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons hold potential for treating Parkinson's disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by coculture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factors produced by stromal cells that result in SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6-conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6-conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell-derived factor-1 α (SDF1α), secreted frizzled-related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified, and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1α, sFRP1, and VEGFD to the culture medium, we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and ßIII-tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1α, sFRP1, and VEGFD are major components of SDIA and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD.

Maternal immune activation by LPS selectively alters specific gene expression profiles of interneuron migration and oxidative stress in the fetus without triggering a fetal immune response.

Maternal immune activation (MIA) is a risk factor for the development of schizophrenia and autism. Infections during pregnancy activate the mother's immune system and alter the fetal environment, with consequential effects on CNS function and behavior in the offspring, but the cellular and molecular links between infection-induced altered fetal development and risk for neuropsychiatric disorders are unknown. We investigated the immunological, molecular, and behavioral effects of MIA in the offspring of pregnant Sprague-Dawley rats given an intraperitoneal (0.25 mg/kg) injection of lipopolysaccharide (LPS) on gestational day 15. LPS significantly elevated pro-inflammatory cytokine levels in maternal serum, amniotic fluid, and fetal brain at 4 h, and levels decreased but remained elevated at 24 h. Offspring born to LPS-treated dams exhibited reduced social preference and exploration behaviors as juveniles and young adults. Whole genome microarray analysis of the fetal brain at 4 h post maternal LPS was performed to elucidate the possible molecular mechanisms by which MIA affects the fetal brain. We observed dysregulation of 3285 genes in restricted functional categories, with increased mRNA expression of cellular stress and cell death genes and reduced expression of developmentally-regulated and brain-specific genes, specifically those that regulate neuronal migration of GABAergic interneurons, including the Distal-less (Dlx) family of transcription factors required for tangential migration from progenitor pools within the ganglionic eminences into the cerebral cortex. Our results provide a novel mechanism by which MIA induces the widespread down-regulation of critical neurodevelopmental genes, including those previously associated with autism.

Age and energy intake interact to modify cell stress pathways and stroke outcome.

OBJECTIVE: Age and excessive energy intake/obesity are risk factors for cerebrovascular disease, but it is not known if and how these factors affect the extent of brain damage and outcome in ischemic stroke. We therefore determined the interactions of age and energy intake on the outcome of ischemic brain injury, and elucidated the underlying mechanisms. METHODS: We utilized a novel microchip-based immunoaffinity capillary electrophoresis technology to measure a panel of neurotrophic factors, cytokines, and cellular stress resistance proteins in brain tissue samples from young, middle-aged, and old mice that had been maintained on control or energy-restricted diets prior to middle cerebral artery occlusion and reperfusion. RESULTS: Mortality from focal ischemic stroke was increased with advancing age and reduced by an intermittent fasting (IF) diet. Brain damage and functional impairment were reduced by IF in young and middle-aged mice, but not in old mice. The basal and poststroke levels of neurotrophic factors (brain-derived neurotrophic factor and basic fibroblast growth factor), protein chaperones (heat shock protein 70 and glucose regulated protein 78), and the antioxidant enzyme heme oxygenase-1 were decreased, whereas levels of inflammatory cytokines were increased in the cerebral cortex and striatum of old mice compared with younger mice. IF coordinately increased levels of protective proteins and decreased inflammatory cytokines in young, but not in old mice. INTERPRETATION: Reduction in dietary energy intake differentially modulates neurotrophic and inflammatory pathways to protect neurons against ischemic injury, and these beneficial effects of IF are compromised during aging, resulting in increased brain damage and poorer functional outcome.

Immunoaffinity capillary electrophoresis: a new versatile tool for determining protein biomarkers in inflammatory processes.

Many diseases caused by inflammatory processes can progress to a chronic state causing deterioration in the quality of life and a poor prognosis for long-term survival. To address inflammatory diseases effectively, early detection and novel therapeutics are required. However, this can be challenging, in part because of the lack of early predictive biomarkers and the limited availability of adequate technologies capable of the identification/characterization of key predictive biomarkers present in biological materials, especially those found at picomolar concentrations and below. This review highlights the need for state-of-the art methodologies, with high-sensitivity and high-throughput capabilities, for determination of multiple biomarkers. Although many new biomarkers have been discovered recently, existing technology has failed to successfully bring this advancement to the patient's bedside. We present an overview of the various advances available today to extend the discovery of predictive biomarkers of inflammatory diseases; in particular, we review the technology of immunoaffinity capillary electrophoresis (IACE), which combines the use of antibodies as highly selective capture agents with the high resolving power of capillary electrophoresis. This two-dimensional hybrid technology permits the quantification and characterization of several protein biomarkers simultaneously, including subtle structural changes such as variants, isoforms, peptide fragments, and post-translational modifications. Furthermore, the results are rapid, sensitive, can be performed at a relatively low cost, without the introduction of false positive or false negative data. The IACE instrumentation can have relevance to medical, pharmaceutical, environmental, military, cultural heritage (authenticity of art work), forensic science, industrial and research fields, and in particular as a point-of-care biomarker analyzer in translational medicine.

Validity and reliability of perinatal biomarkers of adiposity after storage as dried blood spots on paper.

OBJECTIVE: To validate use of chip-based immunoaffinity capillary electrophoresis on dried blood spot samples (DBSS) to measure obesity-related hormones. METHODS: Chip-based immunoaffinity capillary electrophoresis was used to measure adiponectin, leptin and insulin in capillary serum and DBSS in pregnant women and infant heelstick at birth and six weeks. Concordance of measurements was determined with Pearson's correlation and Bland-Altman plots. RESULTS: We report high concordance between results obtained from serum and DBSS. CONCLUSIONS: Ease of sample collection and storage makes DBSS an optimal method for use in studies involving neonates and young children, as well as studies conducted in areas where freezer storage is not available.

Crossing the Antarctica: Exploring the Effects of Appetite-Regulating Hormones and Indicators of Nutrition Status during a 93-Day Solo-Expedition.

Future deep space astronauts must maintain adequate nutrition despite highly stressful, isolated, confined and dangerous environments. The present case-study investigated appetite regulating hormones, nutrition status, and physical and emotional stress in a space analog condition: an explorer conducting a 93-day unsupported solo crossing of Antarctica. Using the dried blood spot (DBS) method, the subject drew samples of his blood on a regular basis during the expedition. The DBSs were later analyzed for the appetite regulating hormones leptin and adiponectin. Energy intake and nutritional status were monitored by analysis of albumin and globulin (including their ratio). Interleukin-6 (IL-6) was also analyzed and used as an energy sensor. The results showed a marked reduction in levels of the appetite-reducing hormone, leptin, and the appetite stimulating hormone, adiponectin, during both extreme physical and psychological strain. Nutrition status showed a variation over the expedition, with below-normal levels during extreme psychological strain and levels abutting the lower bounds of the normal range during a phase dominated by extreme physical hardship. The IL-6 levels varied substantially, with levels above the normal range except during the recovery phase. It was concluded that a daily intake of 5058 to 5931 calories seemed to allow recovery of both appetite and nutritional status between extreme physical and psychological hardship during a long Arctic expedition. Furthermore, IL-6 may be a sensor in the muscle-liver, muscle-fat and muscle-brain crosstalk. These results may help guide nutrition planning for future astronaut crews, mountaineers and others involved in highly demanding missions.

Cytokine patterns and survival in haemodialysis patients.

BACKGROUND: Increased pro-inflammatory cytokine levels are associated with decreased survival. We performed factor analyses to determine if pro-inflammatory and anti-inflammatory cytokines in haemodialysis (HD) patients load onto one or two discrete factors and assessed if patients with a specific pattern of high pro-inflammatory cytokines have decreased survival compared to patients with a high anti-inflammatory cytokine pattern. METHODS: We evaluated 231 HD patients and analyzed them based on the three most common cytokine distribution patterns seen: a high pro-inflammatory group, a high anti-inflammatory group and all others. Survival and Cox regression analyses were performed. RESULTS: Factor analyses of individual cytokines showed that they loaded onto a single factor. Sixty-five patients had a pro-inflammatory pattern of high IL-1, IL-6 and TNF-alpha levels and low anti-inflammatory parameters, including IL-2, IL-4, IL-5, IL-12, CH50 and T-cell number. The next most frequent cytokine pattern was found in 20 patients with high levels of anti-inflammatory parameters. The patients with high pro-inflammatory cytokines had decreased survival compared to patients without a characteristic cytokine pattern. CONCLUSIONS: Further research is needed to better define the underlying causes of increased inflammation among end-stage renal disease patients and to apply anti-inflammatory therapies that may mitigate adverse effects on patient outcomes.

Are breast-fed infants more resilient? Feeding method and cortisol in infants.

The effect of feeding method on stress hormone levels in infants is unknown. We studied infants from birth to 1 year and found salivary cortisol 40% higher in breast-fed infants compared with formula-fed infants. The higher cortisol levels among breast-fed children may be involved in the analgesic effect of breastfeeding.

Chip-based immunoaffinity CE: application to the measurement of brain-derived neurotrophic factor in skin biopsies.

A chip-based immunoaffinity CE system has been employed to measure the concentrations of brain-derived neurotrophic factor in human skin biopsies, taken during atopic inflammatory events. The device employs a replaceable immunoaffinity disk to which capture antibodies have been chemically immobilized. Homogenates obtained from micro-dissected human skin samples were injected into the system, where the analyte of interest was captured in the immunoextraction port, thus allowing non-reactive materials to be removed prior to analysis. The captured analyte was labeled in situ with a red-emitting laser dye before being released from the capture antibody, separated by electrophoresis, and the resolved peaks detected by online LIF. Comparison of this chip-based system with conventional immunoassay demonstrated good correlation when analyzing both standards and patient samples. The system was semi-automated resulting in a CE analysis within 1.5 min and a total of circa 5 min. Intra- and inter-assay CV's of 3.85 and 4.19 were achieved with circa 98.8% recovery of brain-derived neurotrophic factor at a concentration of 100 pg/mL. The assay demonstrated clear differences between clinical stages of atopic dermatitis in human patients and could run 10-15 samples per hour. This system holds the potential for being modified to be a portable unit that could be used in clinics and other biomedical screening studies.

Receptor affinity CE for measuring bioactive inflammatory cytokines in human skin biopsies.

A chip-based receptor affinity CE system has been employed to measure the concentrations of bioactive pro-inflammatory cytokines in biopsy materials obtained from human atopic skin lesions. The device employs a replaceable affinity disk to which recombinant cytokine receptors have been chemically immobilized. Homogenates obtained from micro-dissected human skin samples were injected into the system where the bioactive cytokines were captured in the receptor affinity port and labeled in situ with a laser dye. The captured cytokines were released and separated by CE, the resolved peaks being detected and measured by laser-induced fluorescence. When compared with conventional cell-based bioassays, the affinity receptor chip showed reasonable correlation with r(2) values of 0.998 for interferon gamma, 0.994 for IL-6 and 0.991 for tumor necrosis factor alpha. The complete process including cytokine capture, labeling, and analysis took approximately 12.5 min with intra- and inter-assay CVs below 5.3% and recoveries of 84.9-98.4% at the 100 pg/mL concentration in buffer solutions and 84.5-95% in normal human tissue extract. The system could indicate clear differences between the various clinical stages of atopic dermatitis in human patients and could run 4-6 samples per hour. This system, like previous chip-based systems designed in our laboratory, holds the potential for being modified to be a portable unit that could be used in clinics and other biomedical screening studies.

Application of immunoaffinity capillary electrophoresis to the measurements of secreted cytokines by cultured astrocytes.

The ability of the central nervous system (CNS) to act in conjunction with the immune system has been of great interest to both neurobiologists and immunologists. Previous studies have shown that astrocytes can be stimulated, by various peptides, to act as immune regulators within the CNS and release cytokines and chemokines. However, the regulatory mechanism of astrocytes is still poorly understood. Our present study describes a micro-device capable of monitoring the growth and stimulation of 20 astrocytes by vasoactive intestinal peptide. A microdialysis needle was used to collect the secretion by products, which were analyzed by immunoaffinity capillary electrophoresis (ICE) for the secretion of pro-inflammatory cytokines, IL-1alpha, IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha; hemopoietic cytokines, IL-3, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF); and chemokines; regulated upon activation normal T-cell expression sequence (RANTES), macrophage inflammatory protein (MIP)-1alpha and MIP-1beta. Vasoactive intestinal peptide stimulated astrocytes showed an almost immediate release of pro-inflammatory cytokines and chemokines, with an increase over baseline ranging from 3 to 15 fold, while no substantial increase over baseline was observed for hemopoietic cytokines. This system demonstrates the ability to isolate individual cells in a closely controlled environment and identify and quantify their analytes.

Analysis of Inflammatory Mediators in Newborn Dried Blood Spot Samples by Chip-Based Immunoaffinity Capillary Electrophoresis.

A chip-based immunoaffinity capillary electrophoresis (ICE) system has been developed for measuring inflammatory mediators in dried blood samples routinely taken from newborn babies. A defined area of each dried blood spot was removed from the sample card and its contents eluted. The recovered eluates were injected into the chip and the analytes of interest isolated by the immunoaffinity disk within the chip. The captured analytes were labeled in-situ with a red light-emitting laser dye and electro-eluted into the chip separation channel. Electrophoretic separation of all of the analytes was achieved within 2.0 min with quantification of each peak being performed by online LIF detection and integration of each peak area. The degree of accuracy and precision achieved by the chip-based system is comparable to conventional immunoassays and the system is robust enough to be applied to the analysis of clinical samples.

Biochemicals associated with pain and inflammation are elevated in sites near to and remote from active myofascial trigger points.

OBJECTIVES: To investigate the biochemical milieu of the upper trapezius muscle in subjects with active, latent, or absent myofascial trigger points (MTPs) and to contrast this with that of the noninvolved gastrocnemius muscle. DESIGN: We used a microanalytic technique, including needle insertions at standardized locations in subjects identified as active (having neck pain and MTP), latent (no neck pain but with MTP), or normal (no neck pain, no MTP). We followed a predetermined sampling schedule; first in the trapezius muscle and then in normal gastrocnemius muscle, to measure pH, bradykinin, substance P, calcitonin gene-related peptide, tumor necrosis factor alpha, interleukin 1beta (IL-1beta), IL-6, IL-8, serotonin, and norepinephrine, using immunocapillary electrophoresis and capillary electrochromatography. Pressure algometry was obtained. We compared analyte concentrations among groups with 2-way repeated-measures analysis of variance. SETTING: A biomedical research facility. PARTICIPANTS: Nine healthy volunteer subjects. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Preselected analyte concentrations. RESULTS: Within the trapezius muscle, concentrations for all analytes were higher in active subjects than in latent or normal subjects (P<.002); pH was lower (P<.03). At needle insertion, analyte concentrations in the trapezius for the active group were always higher (pH not different) than concentrations in the gastrocnemius muscle. At all times within the gastrocnemius, the active group had higher concentrations of all analytes than did subjects in the latent and normal groups (P<.05); pH was lower (P<.01). CONCLUSIONS: We have shown the feasibility of continuous, in vivo recovery of small molecules from soft tissue without harmful effects. Subjects with active MTPs in the trapezius muscle have a biochemical milieu of selected inflammatory mediators, neuropeptides, cytokines, and catecholamines different from subjects with latent or absent MTPs in their trapezius. These concentrations also differ quantitatively from a remote, uninvolved site in the gastrocnemius muscle. The milieu of the gastrocnemius in subjects with active MTPs in the trapezius differs from subjects without active MTPs.

Demonstration of a direct capture immunoaffinity separation for C-reactive protein using a capillary-based microfluidic device.

C-reactive protein (CRP), a biomarker of inflammation and cardiovascular disease (CVD) risk assessment, was selected as a model antigen to demonstrate a direct labeling/direct capture immunoaffinity separation. The miniaturized device for immunoaffinity chromatography was constructed from two syringe pumps, a gradient mixing microchip, micro-injector with 250nL capillary injection loop, a capillary column, and a diode laser-induced fluorescence detector fitted with a fused-silica capillary flow cell. Monoclonal anti-CRP was biotinylated and attached to 5.0mum streptavidin-coated silica beads to make the solid support for separation columns. CRP in simulated serum matrix was fluorescently labeled in a one-step reaction and directly injected onto the immunoaffinity capillary. The purified antigen was then eluted in an acid gradient and measured. The antibody binding of CRP was evaluated in two physiological buffers, phosphate buffered saline (PBS) and Dulbecco's PBS (DPBS). A quadratic calibration model produced % relative errors of -15.9 to 12.6 for CRP concentration levels ranging from 0.47 to 95.0mug/mL. The accuracy (% difference from nominal) and precision (% relative standard deviation) of replicate injections were within 17.0%. The limit of detection was 57.2ng/mL and chromatographic run times were less than 10min. The instrument design is simple, and potentially portable, while the assay procedure may be modified for other clinically relevant markers by changing the capture antibody.

Low bone mass in premenopausal women with depression.

BACKGROUND: An increased prevalence of low bone mineral density (BMD) has been reported in patients with major depressive disorder (MDD), mostly women. METHODS: Study recruitment was conducted from July 1, 2001, to February 29, 2003. We report baseline BMD measurements in 89 premenopausal women with MDD and 44 healthy control women enrolled in a prospective study of bone turnover. The BMD was measured by dual-energy x-ray absorptiometry at the spine, hip, and forearm. Mean hourly levels of plasma 24-hour cytokines, 24-hour urinary free cortisol, and catecholamine excretion were measured in a subset of women. We defined MDD according to the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition). RESULTS: The prevalence of low BMD, defined as a T score of less than -1, was greater in women with MDD vs controls at the femoral neck (17% vs 2%; P = .02) and total hip (15% vs 2%; P = .03) and tended to be greater at the lumbar spine (20% vs 9%; P = .14). The mean +/- SD BMD, expressed as grams per square centimeters, was lower in women with MDD at the femoral neck (0.849 +/- 0.121 vs 0.866 +/- 0.094; P = .05) and at the lumbar spine (1.024 +/- 0.117 vs 1.043 +/- 0.092; P = .05) and tended to be lower at the radius (0.696 +/- 0.049 vs 0.710 +/- 0.055; P = .07). Women with MDD had increased mean levels of 24-hour proinflammatory cytokines and decreased levels of anti-inflammatory cytokines. CONCLUSIONS: Low BMD is more prevalent in premenopausal women with MDD. The BMD deficits are of clinical significance and comparable in magnitude to those resulting from established risk factors for osteoporosis, such as smoking and reduced calcium intake. The possible contribution of immune or inflammatory imbalance to low BMD in premenopausal women with MDD remains to be clarified.